内皮缩血管肽反义寡核苷酸促进骨髓移植小鼠造血重建

Antisense oligonucleotide targeting Endostatin enhances hematopoiesis reconstitution in BMT mice

目的观察内皮缩血管肽(Endostatin)反义寡核苷酸转染骨髓基质细胞后在骨髓移植(BMT)小鼠骨髓造血恢复过程中的作用。方法以脂质体作为转染载体,转染不同剂量的 FITC 标记的 Endostatin 反义寡核苷酸,荧光倒置显微镜观察转染效率,用流式细胞术检测最佳转染条件下转染率,用 RT-PCR 和 Western blot 方法检测最佳转染条件下 Endostatin 反义寡核苷酸对 BMT 后不同时间点小鼠骨髓基质细胞 Endostatin mRNA 及其蛋白质和血管细胞间黏附分子1(VCAM-1)mRNA 及其蛋白表达水平的影响。实验分为4组:①正常组:未经任何处理组;②BMT 组:空白对照组;③BMT+转染Endostatin 反义寡核苷酸组;④BMT+转染 Endostatin 错义寡核苷酸组。结果①在体外成功地将 En-dostatin 反义寡核苷酸导入骨髓基质细胞,转染率达86%;②以 Endostatin 反义寡核苷酸转染骨髓基质细胞后,BMT 后不同时间点骨髓基质细胞 Endostatin mRNA 及其蛋白表达被显著抑制[Endostatin 的灰度值分别为(0.09±0.03)～(1.44±1.19)和(0.02±0.02)～(0.14±0.05)](P<0.01或P<0.05),表明转染成功;③Endostatin 反义寡核苷酸转染有效促进了 BMT 后不同时间骨髓基质细胞 VCAM-1mRNA 及其蛋白表达[VCAM-1的灰度值分别为(1.60±0.92)～(8.05±0.87)和(0.07±0.02)～(0.67±0.09)](P<0.01或P<0.05);④Endostatin 错义寡核苷酸对 BMT 后不同时间骨髓基质细胞 Endostatin 和 VCAM-1的表达基本无影响(P>0.05)。结论 Endostatin 反义寡核苷酸可降低Endostatin 表达,增强 VCAM-1的表达,从而促进骨髓微血管生成,改善基质细胞与造血细胞之间和细胞外基质与造血细胞之间的联系而影响骨髓造血。

Objective To explore the effect of antisense oligonucleotide targeting endostatin （endostatin-ASON） transfecting bone marrow stromal cells （BMSC） on hematopoiesis reconstitution in BMT mice. Methods Inhibition of endostatin / VCAM-1 protein and mRNA expression was investigated by transfection of antisense oligonucleotide targeting endostatin with confocal microscopy, Western blot and RT-PCR. Bone marrow stromal cells were cultured and divided into 4 groups： group ① without any treatment; group ② BMT only; group ③BMT ＋ endostatin-ASON transfection; group ④BMT ＋ endostatin scrambled sequence transfection. Results ⑤Endostatin-ASON was successfully introduced into BMSC in vitro, and the transfecting rate was 86% ;②After Endostatin-ASON transfected into BMSC, the expression of Endostatin mRNA and its protein on the BMSC was sigifieantly inhibited at different time point after BMT [the grey value of Endotatin was （0.09±0.03） - （ 1.44±1.19 ） and （0.02±0.02 ） - （0.14±0.05 ）, respectively ] （ P 〈0.01 and P〈0.05 ） ;③Transfecting with Endostatin-ASON effectively promoted the expression of VCAM-1 mRNA and its protein on theBMSC [thegray value of VCAM-1 was （1.60 ±0.92） - （8.05±0.87） and （0.07± 0.02 ） - （0.67 ± 0.09 ）, respedively ] （P〈0.01 and P〈0.05 ） ; ④There was no effects of transfecting Endostatin scrambled sequence on the expression of Endostatin and VCAM-1 on the BMSC （P〉0.05 ）. Conclusion Endostatin-ASON could inhibit Endostatin expression and enhance VCAM-1 expression in BMSC af- ter syngeneic-BMT in mice, which might be one of the mechanisms underlying the endostatin-ASON accelerating hematopoiesis reconstitution after allogeneic-BMT.